Project Summary Tuberculosis (TB) continues to be a major global health problem and development of new interventions requires better understanding of the biological mechanisms used by Mycobacterium tuberculosis (Mtb) to cause infection. Mtb must continuously sense and respond its host environment during infection, and cyclic nucleotides are a critical part of this response in Mtb. Cyclic 3',5'-AMP (cAMP) is a universal signal molecule used by both microbial pathogens and their mammalian hosts to sense and respond to environmental cues. cAMP plays central roles in both bacterial virulence gene regulation and modulation of host response by several major bacterial pathogens, including Mtb. cAMP is produced by adenylyl cyclases and degraded by phosphodiesterases (PDEs) to maintain signaling pathway sensitivity and specificity. Mtb encodes numerous adenylyl cyclases and cAMP-binding effector proteins but only one cAMP PDE (Rv0805) has been identified. Preliminary data indicate that Rv0805 is required for Mtb persistence in a murine model of infection, but its mechanism remains unclear. In addition to hydrolyzing cAMP, Rv0805 has robust activity against 2',3'-cAMP and 2',3'-cGMP, which are products of RNA cleavage, and it has been shown to stabilize cell wall function and regulate gene expression on a global level through unknown mechanisms that may not require cyclic nucleotide hydrolysis. The scientific premise for this proposal is that Rv0805 is a multifunctional protein that contributes to TB pathogenesis, but the importance of RV0805's specific activities for Mtb biology, including virulence, are not known. We hypothesize that Rv0805 has important roles in Mtb biology beyond cAMP hydrolysis that may include both gene regulatory and RNA damage responses. Major goals of this proposal are to determine the importance of Rv0805's catalytic activities for Mtb-host interactions, gene regulation and cAMP homeostasis while establishing whether Rv0805 has the potential to engage in RNA repair. This information will provide a critical basis for understanding the mechanisms underlying Rv0805's role in Mtb biology, particularly during infection.